Retinal neovascularization is a major cause of blindness, and therefore improvements in our understanding of the molecular mechanism underlying this disease are needed to provide better options for treatment. This proposal focuses on understanding the role of two membrane-anchored metalloproteases, ADAMs (a disintegrin and metalloprotease) 9 and 15, in pathological neovascularization in the retina. We have shown that adam 15-/- mice have strongly decreased pathological neovascularization in a model for retinopathy of prematurity (ROP model), whereas adam 9-/- mice, and adam 9/15-/- double knockout mice have strongly increased pathological neovascularization compared to wildtype controls. We now wish to understand the mechanism underlying the role of ADAMs 9 and 15 in proliferative retinopathy. Specifically, we will: i) Identify whether the metalloprotease domain or other domains of ADAMs 9 or 15 are critical for their role in pathological neovascularization using "knock-in" mice. The metalloprotease domain of ADAMs 9 and 15 may be involved in cleaving substrate proteins, the disintegrin domain/cysteine-rich region may function in cell-cell and cell matrix interactions, and the cytoplasmic domain may have a role in signaling. We will generate "knock-in" mice to evaluate the contribution of different domains to neovascularization. ii) Search for potential substrates or interacting partners of ADAMs 9 and 15 that may be relevant for pathological neovascularization. The most likely hypothesis is that ADAM 9 and ADAM 15 cleave substrate oroteins with a role in angiogenesis. Protein ectodomain shedding is a well-established functional regulator of proteins such as TNFa, EGF-receptor ligands, and Notch and Delta. We will therefore test whether ADAMs 9 or 15, or other ADAMs cleave molecules with a role in angiogenesis, such as VEGFR-2, Tie-1 and 2, PECAM, VE-cadherin, etc. However, should "knock-in" mice demonstrate that regulation of angiogenesis is not due to the metalloprotease activity of ADAM 9 and 15, we will search for functionally relevant extracellular or cytoplasmic interacting proteins. Since ADAMs 9 and 15 are not required for normal development and adult homeostasis, we anticipate that the proposed studies will provide new targets for the design of drugs that regulate pathological neovascularization without affecting normal angiogenesis.